The Histopathology of Severe Graded Compression in Lower Thoracic Spinal Cord Segment of Rat, Evaluated at Late Post-injury Phase.

Spontaneous recovery of lost motor functions is relative fast in rodent models after inducing a very mild/moderate spinal cord injury (SCI), and this may complicate a reliable evaluation of the effectiveness of potential therapy. Therefore, a severe graded (30 g, 40 g and 50 g) weight-compression SCI at the Th9 spinal segment, involving an acute mechanical impact followed by 15 min of persistent compression, was studied in adult female Wistar rats. Functional parameters, such as spontaneous recovery of motor hind limb and bladder emptying function, and the presence of hematuria were evaluated within 28 days of the post-traumatic period. The disruption of the blood-spinal cord barrier, measured by extravasated Evans Blue dye, was examined 24 h after the SCI, when maximum permeability occurs. At the end of the survival period, the degradation of gray and white matter associated with the formation of cystic cavities, and quantitative changes of glial structural proteins, such as GFAP, and integral components of axonal architecture, such as neurofilaments and myelin basic protein, were evaluated in the lesioned area of the spinal cord. Based on these functional and histological parameters, and taking the animal's welfare into account, the 40 g weight can be considered as an upper limit for severe traumatic injury in this compression model.

Is Innervation of the Neuromuscular Junction at the Diaphragm Modulated by sGC/cGMP Signaling?

We previously reported NO/sGC signaling in the upper respiratory pathway, receiving input from the respiratory neurons of the brainstem to phrenic motoneurons in the C3-C6 spinal cord. In order to assess whether innervation of the neuromuscular junction (NMJ) at the diaphragm is modulated by sGC/cGMP signaling, we performed unilateral 8-day continuous ligation of the phrenic nerve in rats. We examined sGCß1 within the lower bulbospinal pathway (phrenic motoneurons, phrenic nerves and NMJs at the diaphragm) and the cGMP level in the contra- and ipsilateral hemidiaphragm. Additionally, we characterized the extent of phrenic nerve axonal degeneration and denervation at diaphragm NMJs. The results of our study show that continuous 8-day phrenic nerve ligation caused a marked increase in sGCß1 (immunoreactivity and the protein level) in the ipsilateral phrenic motor pool. However, the protein sGCß1 level in the phrenic nerve below its ligation and the cGMP level in the ipsilateral hemidiaphragm were evidently decreased. Using confocal analysis we discovered a reduction in sGCß1-IR boutons/synaptic vesicles at the ipsilateral MNJs. These findings are consistent with the marked axonal loss (∼47%) and significant NMJs degeneration in the ipsilateral diaphragm muscle. The remarkable unilateral decrease in cGMP level in the diaphragm and the failure of EMG recordings in the ipsilateral hemidiaphragm muscle can be attributed to the fact that sGC is involved in transmitter release at the diaphragm NMJs via the sGC-cGMP pathway.

An Accurate Method for Histological Determination of Neural Tissue Loss/Sparing after Compression-Induced Spinal Cord Injury with Optimal Reproducibility.

In addition to behavioral testing, the efficacy of neuroprotective therapies applied after spinal cord injury (SCI) is commonly evaluated by means of histological quantification of spared neural tissue. The primary insult itself, but mainly the pathological processes of secondary injury are the underlying causes of spinal tissue degeneration, the extent of which depends on the injury severity and post-injury time. Under-estimation of tissue loss due to spinal cord shrinkage and subjective evaluation (impeding reproducibility) are substantial factors that negatively affect the final results. Moreover, processing large numbers of stained spinal cord sections is very time-consuming. To overcome the problem, our new quantification approach combines a modified method for predicting the cross-sectional area at the lesion site with semi-automatic measurement of spared neural tissue and cystic cavities, using freely accessible National Institutes of Health (NIH) ImageJ software, with a Java-based image processing program. Based on the histological parameters measured after differing compression-induced SCI and correlated with behavioral outcomes, we can conclude that our new method is relatively fast, accurate, and optimally reproducible.

Hypothermic treatment after computer-controlled compression in minipig: A preliminary report on the effect of epidural vs. direct spinal cord cooling.

The aim of the present study was to investigate the therapeutic efficacy of local hypothermia (beginning 30 min post-injury persisting for 5 h) on tissue preservation along the rostro-caudal axis of the spinal cord (3 cm cranially and caudally from the lesion site), and the prevention of injury-induced functional loss in a newly developed computer-controlled compression model in minipig (force of impact 18N at L3 level), which mimics severe spinal cord injury (SCI). Minipigs underwent SCI with two post-injury modifications (durotomy vs. intact dura mater) followed by hypothermia through a perfusion chamber with cold (epidural t≈15°C) saline, DMEM/F12 or enriched DMEM/F12 (SCI/durotomy group) and with room temperature (t≈24°C) saline (SCI-only group). Minipigs treated with post-SCI durotomy demonstrated slower development of spontaneous neurological improvement at the early postinjury time points, although the outcome at 9 weeks of survival did not differ significantly between the two SCI groups. Hypothermia with saline (t≈15°C) applied after SCI-durotomy improved white matter integrity in the dorsal and lateral columns in almost all rostro-caudal segments, whereas treatment with medium/enriched medium affected white matter integrity only in the rostral segments. Furthermore, regeneration of neurofilaments in the spinal cord after SCI-durotomy and hypothermic treatments indicated an important role of local saline hypothermia in the functional outcome. Although saline hypothermia (24°C) in the SCI-only group exhibited a profound histological outcome (regarding the gray and white matter integrity and the number of motoneurons) and neurofilament protection in general, none of the tested treatments resulted in significant improvement of neurological status. The findings suggest that clinically-proven medical treatments for SCI combined with early 5 h-long saline hypothermia treatment without opening the dural sac could be more beneficial for tissue preservation and neurological outcome compared with hypothermia applied after durotomy.

A Single Dose of Atorvastatin Applied Acutely after Spinal Cord Injury Suppresses Inflammation, Apoptosis, and Promotes Axon Outgrowth, Which Might Be Essential for Favorable Functional Outcome.

The aim of our study was to limit the inflammatory response after a spinal cord injury (SCI) using Atorvastatin (ATR), a potent inhibitor of cholesterol biosynthesis. Adult Wistar rats were divided into five experimental groups: one control group, two Th9 compression (40 g/15 min) groups, and two Th9 compression + ATR (5 mg/kg, i.p.) groups. The animals survived one day and six weeks. ATR applied in a single dose immediately post-SCI strongly reduced IL-1ß release at 4 and 24 h and considerably reduced the activation of resident cells at one day post-injury. Acute ATR treatment effectively prevented the excessive infiltration of destructive M1 macrophages cranially, at the lesion site, and caudally (by 66%, 62%, and 52%, respectively) one day post-injury, whereas the infiltration of beneficial M2 macrophages was less affected (by 27%, 41%, and 16%). In addition, at the same time point, ATR visibly decreased caspase-3 cleavage in neurons, astrocytes, and oligodendrocytes. Six weeks post-SCI, ATR increased the expression of neurofilaments in the dorsolateral columns and Gap43-positive fibers in the lateral columns around the epicenter, and from day 30 to 42, significantly improved the motor activity of the hindlimbs. We suggest that early modulation of the inflammatory response via effects on the M1/M2 macrophages and the inhibition of caspase-3 expression could be crucial for the functional outcome.

Neuroprotective effect of local hypothermia in a computer-controlled compression model in minipig: Correlation of tissue sparing along the rostro-caudal axis with neurological outcome.

This study investigated the neuroprotective efficacy of local hypothermia in a minipig model of spinal cord injury (SCI) induced by a computer-controlled impactor device. The tissue integrity observed at the injury epicenter, and up to 3 cm cranially and caudally from the lesion site correlated with motor function. A computer-controlled device produced contusion lesions at L3 level with two different degrees of tissue sparing, depending upon pre-set impact parameters (8N- and 15N-force impact). Hypothermia with cold (4°C) saline or Dulbecco's modified Eagle's medium (DMEM)/F12 culture medium was applied 30 min after SCI (for 5 h) via a perfusion chamber (flow 2 ml/min). After saline hypothermia, the 8N-SCI group achieved faster recovery of hind limb function and the ability to walk from one to three steps at nine weeks in comparison with non-treated animals. Such improvements were not observed in saline-treated animals subjected to more severe 15N-SCI or in the group treated with DMEM/F12 medium. It was demonstrated that the tissue preservation in the cranial and caudal segments immediately adjacent to the lesion, and neurofilament protection in the lateral columns may be essential for modulation of the key spinal microcircuits leading to a functional outcome. Tissue sparing observed only in the caudal sections, even though significant, was not sufficient for functional improvement in the 15N-SCI model.

The Characterization of AT1 Expression in the Dorsal Root Ganglia After Chronic Constriction Injury.

To clarify the role of Angiotensin II in the regulation of sensory signaling, we characterized the AT1 expression in neuronal subpopulation of lower lumbar dorsal root ganglia under normal conditions and its alteration in neuropathic pain model. The characterization of AT1 expression was done under control and after the chronic constriction injury induced by four loose ligatures of the sciatic nerve representing the model of posttraumatic painful peripheral neuropathy. Major Angiotensin II receptor type was expressed in approximately 43 % of small-sized and 62 % of large-sized neurons in control. The AT1 overexpression after sciatic nerve ligation lasting 7 days was detected predominantly in small-sized AT1 immunoreactive neurons (about 38 % increase). Chronic constriction injury caused a statistically marked increase in number of the small-sized peptidergic (CGRP immunoreactive) neuronal subpopulation expressing AT1 (about 64 %). The subpopulations of AT1-immunoreactive and nonpeptide-containing primary sensory neurons revealed by IB4 binding, tyrosine hydroxylase- and parvalbumin-immunoreactive neurons were not markedly changed. Our results indicate that: (1) the AT1 overexpression after the chronic constriction injury is an important factor in Angiotensin II-potentiated pain perception; (2) Angiotensin II is involved in pathological mechanisms of neuropathic pain and this effect can be mediated perhaps in combination with other neuropeptides synthesized in the primary sensory neurons.

Baclofen or nNOS inhibitor affect molecular and behavioral alterations evoked by traumatic spinal cord injury in rat spinal cord.

BACKGROUND CONTEXT: The loss of descending control after spinal cord injury (SCI) and incessant stimulation of Ia monosynaptic pathway, carrying proprioceptive impulses from the muscles and tendons into the spinal cord, evoke exaggerated α-motoneuron activity leading to increased reflex response. Previous results from our laboratory have shown that Ia monosynaptic pathway is nitrergic. PURPOSE: The aim of this study was to find out whether nitric oxide produced by neuronal nitric oxide synthase (nNOS) plays a role in setting the excitability of α-motoneurons after thoracic spinal cord transection. STUDY DESIGN: We tested the hypothesis that the inhibition of nNOS in α-motoneurons after SCI could have a neuroprotective effect on reflex response. METHODS: Rats underwent spinal cord transection at Th10 level followed by 7, 10, and 14 days of survival. The animals were treated with Baclofen (a gamma aminobutyric acid B receptor agonist, 3 µg/two times per day/intrathecally) applied for 3 days from the seventh day after transection; N-nitro-l-arginine (NNLA) (nNOS blocator) applied for the first 3 days after injury (20 mg/kg per day, intramuscularly); NNLA and Baclofen; or NNLA (60 mg/kg/day, single dose) applied on the 10th day after transection. We detected the changes in the level of nNOS protein, nNOS messenger RNA, and nNOS immunoreactivity. To investigate the reflex response to heat-induced stimulus, tail-flick test was monitored in treated animals up to 16 days after SCI. RESULTS: Our data indicate that Baclofen therapy is more effective than the combined treatment with NNLA and Baclofen therapy. The single dose of NNLA (60 mg/kg) applied on the 10th day after SCI or Baclofen therapy reduced nNOS expression in α-motoneurons and suppressed symptoms of increased reflex activity. CONCLUSIONS: The results clearly show that increased nNOS expression in α-motoneurons after SCI may be pharmacologically modifiable with Baclofen or bolus dose of nNOS blocker.

Repeated Baclofen treatment ameliorates motor dysfunction, suppresses reflex activity and decreases the expression of signaling proteins in reticular nuclei and lumbar motoneurons after spinal trauma in rats.

The interruption of supraspinal input to the spinal cord leads to motor dysfunction and the development of spasticity. Clinical studies have shown that Baclofen (a GABAB agonist), while effective in modulating spasticity is associated with side-effects and the development of tolerance. The aim of the present study was to assess if discontinued Baclofen treatment and its repeated application leads antispasticity effects, and whether such changes affect neuronal nitric oxide synthase (nNOS) in the brainstem, nNOS and parvalbumin (PV) in lumbar α-motoneurons and glial fibrillary acidic protein in the ventral horn of the spinal cord. Adult male Wistar rats were exposed to Th9 spinal cord transection. Baclofen (30mg/b.w.) diluted in drinking water, was administered for 6 days, starting at week 1 after injury and then repeated till week 4 after injury. The behavior of the animals was tested (tail-flick test, BBB locomotor score) from 1 to 8 weeks. Our results clearly indicate the role of nitric oxide, produced by nNOS in the initiation and the maintenance of spasticity states 1, 6 and 8 weeks after spinal trauma. A considerable decrease of nNOS staining after Baclofen treatment correlates with improvement of motor dysfunction. The findings also show that parvalbumin and astrocytes participate in the regulation of ion concentrations in the sub-acute phase after the injury.

Effect of subpressor dose of angiotensin II on pain-related behavior in relation with neuronal injury and activation of satellite glial cells in the rat dorsal root ganglia.

To clarify the role of angiotensin II (Ang II) in the regulation of sensory signaling, we studied the effect of subpressor dose (150 ng/kg/min) of Ang II on pain-related behavior in relation with neuronal injury and activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRGs) after chronic constriction injury (CCI). Systemic continuous delivery of Ang II induced the tactile, heat and cold hyperlagesia, when measured at 7 days ofpost-injury. Blockade of the AT1 receptor with losartan (2.5 mg/kg/day) prevented tactile hyperalgesia and attenuated cold hyperalgesia, but did not affect the response to noxious heat stimulus. A marked increase of large-sized injured primary afferent neurons, detected by ATF3 immunolabeling, was seen in lower lumbar DRGs on ipsilateral side after Ang II treatment. Subpressor dose of Ang II induced an increase of activated SGCs (detected by GFAP immunolabeling) enveloping large-diameter neurons. Our results suggested that Ang II through the AT1 receptor activation is an important regulatory factor in neuropathic pain perception and plays an important role in the injury of large-sized primary afferent neurons and activation of SGCs elicited by the CCI.

The vulnerability of nitrergic neurons to transient spinal cord ischemia: a quantitative immunohistochemical and histochemical study.

Spinal cord ischemia belongs to serious and relatively frequent diseases of CNS. The aim of the present study was to find out the vulnerability of nitrergic neurons to 15 min transient spinal cord ischemia followed by 1 and 2 weeks of reperfusion. We studied neuronal nitric oxide synthase (nNOS) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in structural elements of lumbosacral spinal cord along its rostrocaudal axis. In addition, a neurological deficit of experimental animals was evaluated. Spinal cord ischemia, performed on the rabbit, was induced by abdominal aorta occlusion using Fogarty catheter introduced into the right femoral artery for a period of 15 min. After surgical intervention the animals survived for 7 and 14 days. nNOS-immunoreactivity (nNOS-IR) was measured by immunohistochemical and NADPHd-positivity by histochemical method, and both immunohistochemical and histochemical stainings were quantified by densitometric analyses. Neurological deficit was evaluated according Zivin's criteria. The number of nNOS-IR and/or NADPH-d positive neurons and the density of neuropil were markedly increased in superficial dorsal horn (laminae I-III) after 15 min ischemia and 7 days of reperfusion. However, ischemia followed by longer time of survival (14 days) returned the number of nNOS-IR and NADPH-d positive neurons to control. In the pericentral region (lamina X) containing interneurons and crossing fibers of spinal tracts, than in lamina VII and in dorsomedial part of the ventral horn (lamina VIII) we recorded a decreased number of nNOS-IR and NADPH-d positive neurons after both ischemia/reperfusion periods. In the medial portion of lamina VII and dorsomedial part of the ventral horn (lamina VIII) we observed many necrotic loci. This area was the most sensitive to ischemia/reperfusion injury. Fifteen minute ischemia caused a marked deterioration of neurological function of hind limbs, often developing into paraplegia. A quantitative immunohistochemical and histochemical study have shown a strong vulnerability of nitrergic neurons in intermediate zone to transient spinal cord ischemia.

Spinal cord transection modifies neuronal nitric oxide synthase expression in medullar reticular nuclei and in the spinal cord and increases parvalbumin immunopositivity in motoneurons below the site of injury in experimental rabbits.

Using immunohistochemistry, we detected the expression of neuronal nitric oxide synthase (nNOS) in ventral medullary gigantocellular reticular nuclei and in the lumbosacral spinal cord 10 days after thoracic transection in experimental rabbits. We tried to determine whether neurons located below the site of injury are protected by the calcium binding protein parvalbumin (PV). Changes of nNOS immunoreactivity (IR) in spinal cord were correlated with the level of nNOS protein in dorsal and ventral horns. Ten days after transection, nNOS was upregulated predominantly in lateral gigantocellular nuclei. In the spinal cord, we revealed a significant increase of nNOS protein in the dorsal horn. This is consistent with a higher density of punctate and fiber-like immunostaining for nNOS in laminae III-IV and the up-regulation of nNOS-IR in neurons of the deep dorsal horn. After surgery, the perikarya of motoneurons remained nNOS immunonegative. Contrary to nNOS, the PV-IR was upregulated in α-motoneurons and small-sized neurons of the ventral horn. However, its expression was considerably reduced in neurons of the deep dorsal horn. The findings indicate that spinal transection affects nNOS and PV in different neuronal circuits.

The impact of short-lasting repeated vibrations on retrograde axonal transport, the expression of CGRP and parvalbumin in lower lumbar dorsal root ganglia.

A prolonged exposure to vibration stimuli triggers pathological changes with many later manifested symptoms. Early vibration-induced changes are still not very well explored. Therefore, short 30 min vibration period per day with frequency 60 Hz repeated for 10 days was used, and the retrograde axonal transport from the sciatic nerve, the expression of calcitonin gene-related peptide (CGRP) and parvalbumin (PV) were studied in the dorsal root ganglia (DRGs) corresponding to lower lumbar spinal levels. Repeated vibration markedly decreased (25 and 34%) the accumulation of retrogradely transported Fluorogold to spinal motor neurons, whereas a significant increase (35 and 25%) was seen in the DRG primary sensory neurons corresponding to the L4 and L5 spinal level. Immunohistochemical studies showed a significant reduction of CGRP-positive small-sized neuronal cells in both DRGs. Fluoro-Jade labeling revealed that marked loss of CGRP-imunoreactive DRG sensory neurons is not due to neuronal degeneration. CGRP protein expression determined by Western blot analysis and optical density measurement, and NGF level measured by ELISA have been decreased, markedly only at the L4 DRG. PV protein expression was not affected by short repeated vibrations. Our results indicate that (a) short-lasting repeated vibrations affect the retrograde axonal transport in the DRG sensory neurons differently than in spinal motor neurons; (b) a decreased NGF-dependent CGRP production in the DRG primary sensory neurons plays an important role in early vibration-induced pathological mechanisms.

Identification of NO/sGC signalling in the bulbospinal respiratory pathway after C2-C3 hemisection of the spinal cord in rats.

Guanylyl cyclase (GC) as the effector molecule for nitric oxide (NO) plays a key role in the NO/cGMP signalling cascade. Based on these observations, our study focused on NO/sGC signalization in the bulbospinal respiratory pathway. The distribution of neuronal nitric oxide synthase (nNOS), ß1 subunit of soluble guanylyl cyclase (ß1sGC) and synaptophysin (SYN) was explored in the upper part of the respiratory pathway after C2-C3 hemisection of the spinal cord in male Wistar rats. Unilateral injection of Fluorogold into the phrenic nucleus (PN) at C4 level and survival of animals for 2 days revealed many Fluorogold fluorescent neurons in the ventral respiratory group (VRG) of the medulla, mostly on the contralateral side. Under physiological conditions we noted nNOS-fluorescent terminals of VRG neurons around ß1sGC fluorescent motoneurons in the PN. A strong depletion of nNOS/SYN fluorescent terminals was noted 8 days after hemisection around alpha motoneurons in the PN on the contralateral side. On the side of injury, nNOS/SYN fluorescent puncta were detected around phrenic motoneurons only sporadically. Phrenic alpha motoneurons responded to C2-C3 hemisection by a loss of ß1sGC positivity. The results confirm, that ß1sGC immunoreactive phrenic motoneurons are innervated by nNOS positive terminals coming from the VRG.

Candesartan reduces the innate immune response to lipopolysaccharide in human monocytes.

OBJECTIVE: Inhibition of angiotensin II receptor type 1 (AT1) reduces chronic inflammation associated with hypertension. We asked whether AT1 receptor inhibition would reduce the innate inflammatory response induced by bacterial lipopolysaccharide (LPS). METHODS: We used unstimulated human circulating monocytes obtained from healthy donors by counterflow centrifugal elutriation. Monocytes were studied in vitro after incubation with LPS (50 ng/ml) with and without 1 mumol/l candesartan, an AT1 receptor blocker. Angiotensin II receptor mRNA expression was determined by reverse transcriptase-PCR and receptor binding by autoradiography; inflammatory factor mRNA expression was studied by reverse transcriptase-PCR and cytokine release by ELISA. RESULTS: Human monocytes did not express detectable AT1 receptors, and angiotensin II did not induce inflammatory factor mRNA expression or cytokine release. However, candesartan substantially reduced the LPS-induced expression of the mRNAs for the LPS recognition protein cluster of differentiation 14, the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1 beta and interleukin-6 and the lectin-like oxidized low-density lipoprotein receptor. In addition, candesartan reduced the activation of the nuclear factor kappa B pathway, the tumor necrosis factor alpha and interleukin-6 secretion, and the ROS formation induced by LPS, without affecting the secretion of interleukin-10. CONCLUSION: We hypothesize that the anti-inflammatory effects of candesartan in these cells are likely mediated by mechanisms unrelated to AT1 receptor blockade. Our results demonstrate that candesartan significantly reduces the innate immune response to LPS in human circulating monocytes. The anti-inflammatory effects of candesartan may be of importance not only in hypertension but also in other inflammatory disorders.

Increased angiotensin II AT1 receptor mRNA and binding in spleen and lung of AT2 receptor gene disrupted mice.

To clarify the relationship between Angiotensin II AT(1) and AT(2) receptors, we studied AT(1) receptor mRNA and binding expression in tissues from AT(2) receptor gene disrupted (AT(2)(-/-)) female mice, where AT(2) receptors are not expressed in vivo, using in situ hybridization and quantitative autoradiography. Wild type mice expressed AT(1A) receptor mRNA and AT(1) receptor binding in lung parenchyma, the spleen, predominantly in the red pulp, and in liver parenchyma. In wild type mice, lung AT(2) receptors were expressed in lung bronchial epithelium and smooth muscle, and were not present in the lung parenchyma, the spleen or the liver. This indicates that AT(1) and AT(2) receptors were not expressed in the same cells. In AT(2)(-/-) mice, we found higher AT(1A) receptor mRNA and AT(1) receptor binding in lung parenchyma and in the red pulp of the spleen, but not in the liver, when compared to littermate wild type controls. Our results suggest that impaired AT(2) receptor function upregulates AT(1) receptor transcription and expression in a tissue-specific manner and in cells not expressing AT(2) receptors. AT(1) upregulation explains the increased sensitivity to Angiotensin II characteristic of the AT(2)(-/-) phenotype, consistent with enhanced AT(1) receptor activation in a number of tissues.

In vivo Angiotensin II AT1 receptor blockade selectively inhibits LPS-induced innate immune response and ACTH release in rat pituitary gland.

Systemic lipopolysaccharide (LPS) administration induces an innate immune response and stimulates the hypothalamic-pituitary-adrenal axis. We studied Angiotensin II AT(1) receptor participation in the LPS effects with focus on the pituitary gland. LPS (50 microg/kg, i.p.) enhanced, 3h after administration, gene expression of pituitary CD14 and that of Angiotensin II AT(1A) receptors in pituitary and hypothalamic paraventricular nucleus (PVN); stimulated ACTH and corticosterone release; decreased pituitary CRF(1) receptor mRNA and increased all plasma and pituitary pro-inflammatory factors studied. The AT(1) receptor blocker (ARB) candesartan (1mg/kg/day, s.c. daily for 3 days before LPS) blocked pituitary and PVN AT(1) receptors, inhibited LPS-induced ACTH but not corticosterone secretion and decreased LPS-induced release of TNF-alpha, IL-1beta and IL-6 to the circulation. The ARB reduced LPS-induced pituitary gene expression of IL-6, LIF, iNOS, COX-2 and IkappaB-alpha; and prevented LPS-induced increase of nNOS/eNOS activity. The ARB did not affect LPS-induced TNF-alpha and IL-1beta gene expression, IL-6 or IL-1beta protein content or LPS-induced decrease of CRF(1) receptors. When administered alone, the ARB increased basal plasma corticosterone levels and basal PGE(2) mRNA in pituitary. Our results demonstrate that the pituitary gland is a target for systemically administered LPS. AT(1) receptor activity is necessary for the complete pituitary response to LPS and is limited to specific pro-inflammatory pathways. There is a complementary and complex influence of the PVN and circulating cytokines on the initial pituitary response to LPS. Our findings support the proposal that ARBs may be considered for the treatment of inflammatory conditions.

Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia.

To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigeminal ganglia, we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of Ang II and substance P in the rat and human trigeminal ganglia. The rat trigeminal ganglia expressed substantial amounts of Ang-N- and ACE mRNA as determined by quantitative real time PCR. Renin mRNA was untraceable in rat samples. Cathepsin D was detected in the rat trigeminal ganglia indicating the possibility of existence of pathways alternative to renin for Ang I formation. In situ hybridization in rat trigeminal ganglia revealed expression of Ang-N mRNA in the cytoplasm of numerous neurons. By using immunocytochemistry, a number of neurons and their processes in both the rat and human trigeminal ganglia were stained for Ang II. Post in situ hybridization immunocytochemistry reveals that in the rat trigeminal ganglia some, but not all Ang-N mRNA-positive neurons marked for Ang II. In some neurons Substance P was found colocalized with Ang II. Angiotensins from rat trigeminal ganglia were quantitated by radioimmunoassay with and without prior separation by high performance liquid chromatography. Immunoreactive angiotensin II (ir-Ang II) was consistently present and the sum of true Ang II (1-8) octapeptide and its specifically measured metabolites were found to account for it. Radioimmunological and immunocytochemical evidence of ir-Ang II in neuronal tissue is compatible with Ang II as a neurotransmitter. In conclusion, these results suggest that Ang II could be produced locally in the neurons of rat trigeminal ganglia. The localization and colocalization of neuronal Ang II with Substance P in the trigeminal ganglia neurons may be the basis for a participation and function of Ang II in the regulation of nociception and migraine pathology.

Anti-inflammatory effects of angiotensin receptor blockers in the brain and the periphery.

In addition to regulating blood pressure, angiotensin II (Ang II) exerts powerful pro-inflammatory effects in hypertension through stimulation of its AT(1) receptors, most clearly demonstrated in peripheral arteries and in the cerebral vasculature. Administration of Ang II receptor blockers (ARBs) decreases hypertension-related vascular inflammation in peripheral organs. In rodent models of genetic hypertension, ARBs reverse the inflammation in the cerebral microcirculation. We hypothesized that ARBs could be effective in inflammatory conditions beyond hypertension. Our more recent studies, summarized here, indicate that this is indeed the case. We used the model of systemic administration of the bacterial endotoxin lipopolysaccharide (LPS). LPS produces a robust initial inflammatory reaction, the innate immune response, in peripheral organs and in the brain. Pretreatment with the ARB candesartan significantly diminishes the response to LPS, including reduction of pro-inflammatory cytokine release to the general circulation and decreased production and release of the pro-inflammatory adrenal hormone aldosterone. In addition, the ARB very significantly decreased the LPS-induced gene expression of pro-inflammatory cytokines and microglia activation in the brain. Our results demonstrate that AT(1) receptor activity is essential for the unrestricted development of full-scale innate immune response in the periphery and in the brain. ARBs, due to their immune response-limiting properties, may be considered as therapeutically useful in a number of inflammatory diseases of the peripheral organs and the brain.